The present invention relates generally, in one embodiment, to the preparation of biological samples. For example, the present invention, in one embodiment, relates to a system and method for the automated, continuous work flow, pre-treatment and processing of biological samples.
In this disclosure, the term “staining” is used to refer to the end product of a process, by which certain parts of the sample may be stained, i.e. have obtained a different color, either in the optic range or in another electromagnetic range, such as ultra violet, or the staining may be a detectable (e.g., automatically detectable) change in properties, such as fluorescent properties, magnetic properties, electrical properties or radioactive properties. To obtain the staining, the sample may undergo a series of treatment steps, such as—but not limited to—washing, binding of reagents to the specific parts of the sample, application of the reagents, etc. and each treatment step may include a plurality of individual treatments.
Examples of sample preparation and processing that may be used in the practice of the invention include but are not limited to the following.
Sample processing in immunohistochemical (IHC) applications and in other chemical and biological analyses may involve one or a number of various processing sequences or protocols as part of an analysis of one or more samples. The sample processing sequences or protocols may be defined by the individual or organization requesting an analysis, such as a pathologist or histologist of a hospital, and may be further defined by the dictates of a particular analysis to be performed.
In preparation for sample analysis, a biological sample may be acquired by known sample acquisition techniques and may comprise, for example, in IHC applications, tissues generally or, even, in some applications one or a plurality of isolated cells, such as in microarray samples, and may be presented on a microscope slide or a similar plane, rectangular sample carrier. Furthermore, the sample may be presented on the slide or other carrier variously and potentially in some form of preservation. As one example, a sample such as a layer or slice of skin may be preserved in formaldehyde and presented on a slide with one or more paraffin or other chemical layers overlying the sample. Samples preserved with paraffin may need to undergo deparaffinization, a process by which paraffin layers overlaying the sample are removed. In addition, the target or sample may need to be restored to a condition where it is suitable for staining operations—a process known as target retrieval.
Immunologic applications, for example, may involve processing sequences or protocols that comprise steps such as deparaffinization, target retrieval, and staining, especially for in-situ hybridization (ISH) techniques. Previously, in some applications, these steps may have been performed manually, potentially creating a time-intensive protocol and necessitating personnel to be actively involved in the sample processing. Attempts have been made to automate sample processing to address the need for expedient sample processing and a less manually burdensome operation. However, such previous efforts may have not fully addressed the needs for an automated sample processing system. Some prior efforts to automate sample processing may be deficient in several aspects that prevent more robust automated sample processing, such as: the lack of sufficient computer control and monitoring of sample processing; the lack of information sharing for processing protocol and processing status, especially for individual samples; the lack of diagnostic capabilities; and the lack of real-time or adaptive capabilities for multiple sample group processing. At least one of these may be found in embodiments of the present invention.
The staining procedure may be laborious and use many different reagents. The staining protocol may include the following steps: deparaffinization, washing, antigen retrieval, endogenous biotin or enzyme blocking, incubation with immunological reagents, molecular probes, secondary visualization reagents and various chromogen reagents, washing steps and counterstaining.
Certain methods for mixing reagents and liquids have been in practice, for example, in the context of mixing processes for staining instruments such as IHC and ISH equipment. However, in some applications, such mixing processes have been cumbersome. Thus, some existing systems may have disadvantages in their implementation of mixing processes, for example, due to the complex use of reagents in the staining procedure. Below, a few non-limiting examples are listed in more detail. These examples may be useful and applied to the practice of the invention.
The chromogen reagents (e.g. DAB, AEC, fast red etc) often come as concentrated reagents in organic or high viscosity solutions and need to be diluted prior to being applied to the sample. Chromogens like the Fast Red alkaline phosphatase chromogen are made ready for use by mixing and dilution of two or three reagents, which are very different in nature with regard to salt content, viscosity and density. Furthermore, the resulting mixtures are unstable over time and need to be used within a short time. Some chromogens suitable for e.g. horseradish peroxidase, like DAB and AEC, are easily oxidized when exposed to air during e.g. vigorous mixing or dilution.
The enzyme chromogens and counter-stain reagents (e.g., hematoxylin) are semi oxidized and can contain precipitates and solids. By further oxidation or slight change in pH, the reagents can further precipitate.
Antibody and enzyme containing reagents often contain stabilizing proteins and or detergents, which cause the solution to foam when being shaken or stirred. Many proteins cannot easily tolerate to be exposed to the hydrophobic air in foam. Wash buffers can contain detergents, which can foam when shaken or stirred. The foam can spread to other compartments of the instrument in an unwanted and unpredictable way. Mixing of some reagents (e.g. the HRP chromogens and peroxide reagents) can result in the formation of small bubbles. These can generate foam or bubbling on the surface of the mixture.
Spill over/carry over is often undesirable. The staining process may be characterized by using many, complex and very different reagents and buffers and in many different dilution ratios and mixtures. Some of the reagents or buffers are incompatible with each other. In the event of cross contamination due to e.g. carry over, the reagents may be ruined within seconds or solids can precipitate, making the staining unsuccessful. For example, enzyme containing reagents can not be mixed with the corresponding chromogens, or high salt concentrates may not be mixed with e.g. proteins containing mixtures, or organic solvents can not be mixed with protein containing mixtures, or highly pH buffered wash buffers can not be mixed with low buffered mixtures without significantly altering the properties of the reagents. Accordingly a need exists for cleansing and washing of the mixing device in an efficient manner.
As some procedures may be regarded as complex, existing instruments may use many different protocols resulting in less predictable results due to even minute amounts of reagent carry-over or unplanned mixing of reagents. Reagent mixing efficiency may be, in part, affected by a software protocol and how it attempts to group an application of reagent mixtures on sample carriers such as slides so as to maximize efficiency, for instance, by minimizing reagent waste. Consequently, it may be desired for a mixing device for an automatic biological sample processing apparatus ideally to be very efficient and to be designed for a variety of reagent mixing protocols and sequences. It may be further desirable to provide on-demand mixing of reagents within a continuous processing environment.
During staining, build-up of small fouling layers on the various surfaces may cause problems, as the typical staining protocol calls for many mixing and dilution steps. Consequently, in one embodiment of the invention, the mixing device may have a minimum of surface area and very smooth surfaces. Furthermore, the mixing device may ideally be able to mix very different volumes of reagents in both small and large volumes ratios. In other words, the degree of dilution and mixing ratios of reagents may vary from small to high ratios. In summary, the mixing device in one embodiment, may ideally allow at least one of: mixing of small and large volumes; mixing reagents with different viscosities and densities; mixing of immiscible or nearly immiscible reagents; no fouling of mixing rods or similar due to precipitated material; easy escape of formed gasses during mixing; prevention of foaming of e.g. detergent or protein containing reagents; low build-up of debris or fouling on the device surfaces; easy emptying and washing—regardless of volumes; and very low reagent carry-over. At least some present mixing systems for automated biological sample processing do not truly fulfill one or more of the above-mentioned properties. On-the-slide mixing may not allow for very large ratios of dilution. It may also not allow for efficient mixing of reagents with very different densities or viscosities. In one embodiment, on-slide mixing is used as an alternative means to reagent mixing, for example, as described in U.S. patent application Ser. No. 11/177/730, filed Jul. 8, 2005, the disclosure of which is hereby incorporated by reference.
Further, a defined staining protocol, for use in the invention, may include one or more defined temperatures. For many IHC applications, and many sample processing sequences and protocols, it may be desired generally to have temperature characteristics associated with the sample, sample carrier, and the processing environment. Traditional sample processing technology, which also may be used in the practice of the invention, has provided temperature control through heating devices that heat an entire set of sample carriers in the sampling processing system. Other technologies, such as the sample processing system described in U.S. Pat. No. 6,183,693, may provide heating devices for individual sample carriers that are individually controlled to heat the slides. However, each of these traditional sample processing systems may lack a desired degree of temperature control or temperature tolerances.
Inadequacies in temperature control of traditional technologies may include uncontrolled cooling. Traditional systems may only provide ambient cooling when the heating devices are off. Ambient cooling is not considered active control and may not meet protocol temperature requirements or may not otherwise be optimal. Although heating and heat control may be features of such systems, controlled cooling of the samples, sample carriers, and processing environments may not always be adequately addressed. Cooling techniques such as hooded fans may be incorporated in some traditional technologies. However, these devices can lack sufficient capabilities of temperature control to meet certain protocol requirements, especially temperature tolerances for samples, sample carriers, reagents, and ambient system temperature.
Traditional systems may even lack temperature control, perhaps as related to temperature tolerances generally, as such tolerances may not be adequately maintained during ambient or other traditional cooling, or during processing sequences or events, generally. In some protocols, for example, the temperature tolerances during non-heating periods may be such that uncontrolled temperature changes may produce undesirable results during the processing sequence. Other IHC processes of the protocol may be adversely affected by uncontrolled temperature changes, the degree of temperature change, and temperature changes outside of acceptable tolerances. The lack of temperature control may actually dissuade technologists from employing automated processing sequences or protocols, especially IHC sequences, that may be dependent upon a particular temperature tolerance and the amount of temperature change during a processing sequence.
Certain types of temperature control may not have even been addressed in traditional sample processing system technologies. In one embodiment of the present invention, reagent temperatures are controlled. Reagents can play a vital role in the staining sequence of many processing protocols. The quality of the reagents, therefore, may be important for adequate sample processing. Reagents, for example, can have a certain shelf life that may be limited if maintained at undesirable temperatures such as the typical ambient temperatures of traditional biological sample processing systems and the laboratories housing such systems. Traditional technologies may lack the temperature control needed to optimally preserve the reagents stored in the processing system that are often subject to inadequate or changing ambient temperatures of such systems and the laboratory environment.
In one embodiment, sample processing apparatuses for staining and treating samples by means of probes may comprises a first section or station for containing one or more reagent containers, such as bottles or vials; a second section or station for mounting slides, a probe arranged to aspirate a portion of reagent from a selected reagent container and dispensing the reagent to a slide on which the sample is arranged and a drive means for moving the probe between the various sections.
Past efforts at automated sample processing for samples presented on carriers such as slides, such as U.S. Pat. No. 6,352,861 and U.S. Pat. No. 5,839,091, have not afforded at least some of the advantages and other combinations of features as presented herein. U.S. Pat. No. 5,948,359 discloses an apparatus of the above mentioned type, wherein the first station comprises a vial holder for holding 40 or more vials in order to provide a wide range of different reagents adapted for different staining purposes, and thereby the possibility of automatically staining a large number of slides requiring different staining processes. In practice it is often desired that the apparatus facilitates that many different staining processes can be performed at the same time in the apparatus, because this avoids the necessity of grouping samples requiring the same procedure or other treatment with reagents, and processing each group individually.
Currently available biological staining apparatuses do not provide for sample pre-treatment. In one embodiment of the present invention, a staining apparatus provides for pre-treatment. Biological samples, such as tissue samples, are usually prepared before the staining can be performed. The tissue slides are subjected to a pre-treatment process depending upon the type of staining process that is to be performed on the tissue. This pre-treatment could include deparaffinization or target retrieval. The preparation of the tissues on the slides is often carried out manually in the laboratory before they are loaded into the automatic staining instrument. This pre-treatment includes immersing the slide, for example, in one or more buffers or other types of processing liquid for a predetermined amount of time and temperature. Immersion processes may include multiple processing liquids such as in a specified sequence of processing liquids such as in a specified sequence of processing liquids. Unfortunately, however, this manual preparation may be cumbersome and the pre-treatment may be insufficient, since the amount of time and the temperature of the liquid should be observed substantially precisely in order to achieve the correct pre-treatment result. For example, pre-treatment may consist of multiple-step deparaffinization sequence of fluids that remove paraffin used to preserve tissue samples and/or heat-induced antigen retrieval which consists of immersion in or more fluids for specific periods at specific temperatures. Preparation of the aforementioned pre-treatment processes and the like may be difficult to achieve via manual preparation.
In the U.S. Pat. No. 5,839,091, an automated staining apparatus is disclosed wherein a plurality of biological samples accommodated on microscope slides may be processed. However this instrument does not provide a processing tank for pre-treatment of the slides.
Some staining processes involve the use of hazardous materials, such as toxic materials. These materials may be collected in special containers in order to ensure safe handling of the waste material. However, this does not sufficiently protect the laboratory environment in which the apparatus is placed from being contaminated with toxic material. Moreover, in some staining processes or other treatments in the apparatus, heat is applied. This increases the risk of vaporizing reagents which then may escape to the outside of the apparatus.
In some apparatuses, a protective hood or similar plastic cover have been put over the staining apparatus in order to shield off the biological samples during the staining. In this known technique, one may risk the drying out of slides and lack of control of airspeed and temperature.
Some staining apparatuses require existing sequences to finish before inserting or changing the aggregate in some manner. In one embodiment of the invention, a staining apparatus is provided that is capable of providing pre-treatment processes, for example, to carriers containing samples which may be conducted independently and simultaneously with processing other carriers containing samples that are also undergoing pre-treatment or staining sequences asynchronously. In addition, operators often have needed particular knowledge and skills in order to assure the integrity of the process, the instrument, or the result. Thus, one embodiment of the invention reduces such effects by providing a system which is more user, operator, supplier, and/or manufacturer friendly and adaptable to real-world conditions and events.
Hence, some conventional apparatuses hitherto have not provided for adequate sample pre-treatment. Biological samples, such as tissue samples, are usually prepared before the staining can be performed and may be subjected to a pre-treatment process depending upon the type of staining process that is to be performed on the tissue. Pre-treatment processes are generally carried out manually in a laboratory and may include deparaffinization or target retrieval. In addition, pre-treatment processes may also require immersion of the slide in a buffer, or in other types of processing liquids for some predetermined amount of time and at a specific temperature. Manual sample preparation may be cumbersome because pre-treatment steps are often subject to stringent constraints and are sensitive to minute variations in experimental conditions. Consequently, small deviations in the pre-treatment protocol may lead to insufficient pre-treatment and inaccurate results.
Thus, there is a need for systems and methods to allow for the automatic real-time continuous processing of biological samples, so that once a carrier containing a sample, such as a slide, has been prepared and introduced into an apparatus, it is processed in accordance with specified protocols, in conformity with any constraints, and, with minimal, or in some cases, with no further user-intervention.
There is also a need for systems that automate the sequencing of sample processing to maximize throughput and that allow users to track and monitor the status of slides in the apparatus. Additionally, on account of the sensitive nature of the process, there is a need to provide feedback to users about processing related errors, or a lack of resources in sufficient time for corrective action to be taken. Moreover, there is a need to collect both slide and apparatus related information and share the collected information so as to improve efficiency and allow automatic interaction with other information processing systems.
At least one of the foregoing needs are met, to a great extent, by at least one embodiment of the disclosed invention, wherein in one aspect the present disclosure relates, in part, to the field of software and hardware for the control, management, tracking, monitoring, scheduling and diagnosing of automatic biological sample processing systems. In some embodiments, systems, methods and apparatus allow for the automatic pre-treatment of the biological samples on slides or other similar carriers or substrates in an automatic staining apparatus so that the entire processing of the biological samples may be performed automatically in a single physical apparatus.
In accordance with one aspect of the present invention, a method of processing a plurality of biological samples in an apparatus is provided that in some embodiments provides applying at least one reagent from a plurality of reagents to at least one first biological sample according to a sequence of steps in a first protocol, wherein at least one second biological sample can be added or removed from the apparatus prior to completion of the first protocol, and wherein at least one second reagent can be added or removed from the apparatus prior to completion of the first protocol.
In accordance with another aspect of the present invention, a method of processing a plurality of biological samples in an apparatus is provided that in some embodiments provides applying at least one reagent from a plurality of reagents to at least one first biological sample according to a sequence of steps in a first protocol. The method may also include, prior to completion of the first protocol, adding a second biological sample to the apparatus and applying at least one reagent from the plurality of reagents to the second biological sample according to a sequence of steps in a second protocol.
In accordance with yet another aspect of the present invention, a method of processing a plurality of biological samples in an apparatus is provided that in some embodiments provides applying at least one reagent from a plurality of reagents to at least one first biological sample according to a sequence of steps in a first protocol and applying at least one reagent from a plurality of reagents to at least one second biological sample according to a sequence of steps in a second protocol. The method may also include determining the first-completed biological sample, being whichever one of the first biological sample and the second biological sample whose processing is completed prior to the completion of the processing of the other biological sample and removing the first-completed sample from the apparatus without interrupting the processing of the other biological sample.
In accordance with yet another aspect of the present invention, an apparatus for automated processing of a plurality of biological samples is provided that in some embodiments provides at least one probe for dispensing at least one reagent, a plurality of reagent containers, and a plurality of biological sample carriers wherein processing of each biological sample is conducted according to a respective sequence of protocol steps, wherein a first biological sample, on a first sample carrier, for which the processing is completed may be removed from the apparatus simultaneous with the processing of a second biological sample on a second carrier, and wherein a first reagent container may be removed or replaced simultaneous with the processing of at least one biological sample.
In accordance with yet another aspect of the present invention, an apparatus for automated processing of a plurality of biological samples is provided that in some embodiments provides a means for dispensing at least one reagent and means for individually supporting a plurality of biological samples wherein processing of each biological sample is conducted according to a respective sequence of protocol steps. The apparatus may also include a means for scheduling the order of protocol steps needed for processing a plurality of biological samples, wherein a first biological sample, on a first sample carrier, for which the processing is completed, may be removed from the apparatus simultaneous with the processing of a second biological sample on a second carrier, wherein a first reagent container may be removed or replaced simultaneous with the processing at least one biological sample.
In accordance with yet another aspect of the present invention, an apparatus for automated processing of a plurality of biological samples is provided that in some embodiments provides at least one probe for dispensing at least one reagent, a plurality of reagent containers, and a plurality of biological sample carriers, wherein processing of each biological sample is conducted according to a respective sequence of protocol steps, wherein a first biological sample, on a first sample carrier, for which the processing is completed, may be removed from the apparatus simultaneous with the processing of a second biological sample on a second carrier, and wherein a first reagent container may be removed or replaced simultaneous with the processing at least one biological sample.
There are, of course, additional embodiments of the invention that will be described below.
In this respect, before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.
As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.